Introduction

Mark Mattson led this live discussion on 30 April 2002. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:

Live Discussion held 30 April 2002.

Participants: Mark Mattson, Anthony White, Andrew McCaddon, Eugene Rogers, Tom Shea,Larry Sparks, Keith Crutcher, Gladys Maestre, Amy B. Graves, PS (John), Paul Aisen, Monique Breteler, Ralph Green, Gabrielle Strobel

Note: The transcript has been edited for clarity and accuracy.

Anthony White: Hi Andrew. I think this is an excellent way to exchange ideas. Without forums like this, there are only conferences

Andrew McCaddon: I'm a general practitioner in North Wales, so don't get to many conferences!

Anthony White: That's a shame, given your track record in this area.

Andrew McCaddon: Anthony, where are you based?

Anthony White: I was in Australia until mid-2001 with Colin Masters and Roberto Cappai. I am now at Imperial College in London. My primary area at present is prion research but I am still collaborating with our group on homocysteine and AD

Andrew McCaddon: I wonder if we've miscalculated the time difference between the UK and the US? I always get confused when we move the clocks around here!! We are probably an hour early.

Anthony White: It will be interesting to see if the research in this area is starting to formulate into a coherent mechanism of action.

Andrew McCaddon: It probably is. The hypothesis we will discuss might extend to other neurodegenerative diseases, as well as aging per se.

Anthony White: A unifying hypothesis should at least help to focus research on a more defined process rather than looking for homocysteine effects under every rock. I think this is the state with homocysteine and cardiovascular disease. Every paper has a different angle.

Mark Mattson: {enters} Hello, did everyone take their folate today? According to Tom Shea, we should also be taking vitamin E and avoid eating nails (iron), especially if we harbor an apoE4 allele in our genome.

Eugene Rogers: {enters}

Tom Shea: {enters}

Andrew McCaddon: Hi Tom. Anthony and I have been here chatting for an hour! We miscalculated the time zone!

Mark Mattson: Hey Tom. Glad to have you on board. Andrew, did your pre-chat chat uncover any novel insight into folate, homocysteine and the inner sanctions of the brain?

Andrew McCaddon: I emailed Anthony an abstract of a forthcoming hypothesis paper that I'd like to discuss today. He's gone off to read it over a cup of tea.

Keith Crutcher: {enters} Hello all.

Keith Crutcher: Is that Mark I see over there with a bottle of folic acid in his hand?

Larry Sparks: {enters}

Mark Mattson: Larry, everything is going full bore here. Did you ever finish analyses from the TNF receptor knockout mice on the high cholesterol diet? It was in the last millennium that we began the collaboration!

Larry Sparks: Mark, the TNF mouse experiments could be run again.

Amy B. Graves: {enters} Hi all.

PS: {enters}

Paul Aisen: {enters}

Gabrielle Strobel: {enters} Hi Mark, sorry to be late, I was stuck in the lobby. Hello and welcome everyone. I am Gabrielle Strobel, managing editor of the Alzforum, and will moderate today's discussion. I'd like to start by asking everyone to please type in your full name and state where you are from so we all know who is here.

Mark Mattson: Mark Mattson, National Institute on Aging and Johns Hopkins.

Amy B. Graves: I'm Amy Borenstein Graves, professor in epidemiology and biostatistics at the University of South Florida , doing work in risk factors for AD.

Eugene Rogers: Hello everyone, I am from UMass Lowell.

Tom Shea: Hi. Biology, UMass Lowell. Gene Rogers and I have been studying homocysteine toxicity in culture and folate deprivation in culture and in ApoE knockout mice.

PS: I'm John, a respiratory therapist from Palm Springs, California. My mother just developed AD and I'm next, thus my keen interest in this.

Andrew McCaddon: I'm a general practitioner in North Wales with an interest in B12 deficiency and AD.

Keith Crutcher: I'm at the University of Cincinnati.

Larry Sparks: Hello, I am from the Sun Health Research Institute, Sun City, Arizona. I am running the AD cholesterol-lowering treatment trial.

Paul Aisen: Hello. I am from Georgetown University. I am conducting studies of homocysteine reduction in AD.

Anthony White: Hi, I am at Imperial College, London, looking at interactions between homocysteine and metals and their effects on neurons.

Gabrielle Strobel: Why don't we start by asking our guest of honor, Mark, to restate briefly what he thinks we know now about how homocysteine affects neurons in Alzheimer's and other neurologic conditions.

Mark Mattson: Cell culture and in vivo data from mouse models of AD suggest that homocysteine can directly endanger/damage neurons. It enhanced DNA damage by reducing repair and by promoting oxidative stress. The literature on the role of homocysteine in one-carbon metabolism is consistent with such a mechanism.

Gabrielle Strobel: Just to throw in a brand-new literature update: JAHA later this week will run a paper by Lydia Bazzano et al. showing high dietary folate decreases stroke risk. 9,764 participants in the NHEFS nutrition survey were asked about eating habits 5 times over 10 years. No surprise there (Bazzano et al., 2002).

Gladys Maestre: {enters}

Gabrielle Strobel: Hello Gladys!! Gladys is from the University of Maracaibo, Venezuela, and studies homocysteine in the Maracaibo Study on Aging. Welcome!

Gabrielle Strobel: Mark, the homocysteine-Aβ-DNA damage link is new and intriguing. How could that work? Do you know which repair processes homocysteine interferes with? Does it inhibit any of the known repair DNA enzymes?

Mark Mattson: In our hands homocysteine increases both uracil misincorporation and oxidative DNA base modifications. We are currently determining whether UDGs or FPGs, Ku proteins, etc are affected by homocysteine. We do know that PARP and p53 are players in the cell death cascade induced by homocysteine.

Gabrielle Strobel: Mark, Ku proteins--that is interesting. Do postmitotic neurons have double-strand breaks?

Mark Mattson: Yes, they can have double strand breaks. Also, Ku localizes to telomere ends, and we are finding that (in embryonic neurons) telomerase is a "guardian of the genome" that protects against DNA damage.

Gabrielle Strobel: What happens with a neuron that has double-strand breaks and tries to re-enter the cell cycle, as some are finding. Can it repair these breaks? See our May 20 chat on cell cycle in AD.

Mark Mattson: Gabrielle, the cell cycle re-entry hypothesis is alive and well. Since neurons are already postmitotic, the attempt to re-enter the cell cycle may trigger apoptosis.

Amy B. Graves: To what extent do people feel the homocysteine/folate link with AD is responsible for the cardiovascular association with AD?

Andrew McCaddon: Amy, I think the elevated homocysteine reflects a pathogenic mechanism common to both--namely the effects of oxidative stress on B12 metabolism.

Larry Sparks: Our work has shown that most AD subjects have heart disease and that many individuals with heart disease have AD-like neuropathology. It might be hard to separate AD-heart disease and HC in that vein.

Amy B. Graves: So Andrew, you think the homocysteine link is working independently of the rest of the cardiovascular piece?

Gladys Maestre: Our work in a population sample studied extensively from the cardiovascular disease suggests that the risk for AD is independent of the cardiovascular effect.

Amy B. Graves: Gladys: Can you clarify what you mean with cardiovascular disease and the cardiovascular effect?

Gladys Maestre: The risk of AD conferred by homocysteine is independent. It is still strong even when we account for hypertension, cardiovascular events, history of stroke, lipids, and even blood pressure, circadian dysregulation, arrhythmias.

Amy B. Graves: Gladys and Paul, this is important. Maybe implies different causative mechanisms.

Paul Aisen: Amy, it is consistent with a direct contribution of homocysteine to amyloid neurotoxicity.

Amy B. Graves: Paul, so that means that HC is directly involved in amyloid deposition?

Paul Aisen: No, I think it is more likely that homocysteine increases the sensitivity of neurons to amyloid-mediated damage.

Eugene Rogers: In cell culture, neurotoxicity occurs with high homocysteine independently, of course, because there are no vascular issues.

Andrew McCaddon: Mark, we have a paper in the next edition of "Neurology" presenting a hypothesis complementary to yours, namely that oxidative stress might contribute to elevated serum homocysteine (McCaddon et al., 2002; see also Cobalaminergic Hypothesis.)

Mark Mattson: Andrew, your findings make sense and would fit with Tom's studies in ApoE KO mice, which seem to have decreased antioxidant capacity.

Tom Shea: If we co-treated with S-adenosyl methionine, homocysteine-induced apoptosis was prevented--depletion of essential methylation of DNA due to homocysteine accumulation?

Mark Mattson: One interesting question is: under conditions of folate deficiency do neurons themselves generate the homocysteine that may eventually kill them? Does folate deficiency have different effects on homocysteine production in different cell types?

Eugene Rogers: Folate deficiency has different effects on different cell types. E.g. transulfuration and betaine-homocysteine methyl transferase pathways are essentially absent in neurons, so homocysteine removal is basically dependant on remethylation to methionine with methylene tetrahydrofolate reductase and methionine synthase, or on cellular export, where it may accumulate in the extracellular fluid or the CNS.

Anthony White: We have also found that increases in oxidative stress or a reduction in antioxidant levels in cultured neurons makes them highly vulnerable to homocysteine. This would fit with Andrew's theory.

Mark Mattson: Alterations in lipid (shingolipids and cholesterol) metabolism and oxidative stress are common to many different age-related diseases, including cardiovascular and Alzheimer's. My take on this is that these alterations might differentially occur in blood vessels versus neurons, but more often occur simultaneously.

Paul Aisen: The paper from the Oxford group published a few years back in Archives of Neurology showed a strong link between homocysteine levels and neuropathologically pure AD (Clarke et al., 1998). That is, the homocysteine-AD link seems to be independent of vascular disease.

Andrew McCaddon: We also now have some interesting data accepted showing elevated serum cysteine in our AD patients, and a correlation between cognitive scores and total serum glutathione ( see abstract). Our interpretation was that we are seeing the effects of oxidative stress on methionine synthase and cystathionine beta-synthase activity.

Larry Sparks: Was there a negative relationship between glutathione levels and cognitive scores and what instruments were used?

Anthony White: Andrew, loss of glutathione in our neurons increases toxicity of homocysteine and cysteine (the latter to a lesser extent).

Eugene Rogers: Anthony, do you first observe an initial increase in neuronal glutathione before depletion and then enhanced toxicity to homocysteine and cysteine?

Anthony White: Gene, we haven't tested the full time course, but sub-toxic levels of homocysteine don't appear to alter glutathione levels in our neurons early on.

Andrew McCaddon: All: Our "functional" vitamin B12 deficiency hypothesis requires in vitro testing. Anyone interested in running a tissue culture experiment for us?

Mark Mattson: Andrew, we could address the B12 issue in our hippocampal cultures.

Andrew McCaddon: Yes! Excellent, Mark. What you need to do is culture those neurons in a homocysteine-free medium, and then "oxidatively stress" them--they should pump the stuff out into the medium.

Tom Shea: Homocysteine does synergistically enhance Aβ-??????? ROS and apoptosis (Ho et al., 2002).

Monique Breteler: {enters}

Gabrielle Strobel: Welcome, Dr. Breteler!!

Monique Breteler: Hi all. I have to figure out how to work this.

Larry Sparks: You're doing fine, buddy.

Gabrielle Strobel: We've got England, the US, the Netherlands, and Venezuela all together here. Monique, you have figured out all there is to it. Type in the white box and hit return.

Monique Breteler: Ok, thanks.

Gladys Maestre: Homocysteine really increments with age and is higher in men but there are no real data on why this might be--any thoughts?

Mark Mattson: Since oxidative stress increases with age, one could propose that the increased homocysteine levels are secondary to oxidative stress. Folate levels also tend to decrease with age, so there could be a diet/absorption/conversion problem during aging.

Tom Shea: We saw a large increase in homocysteine in culture medium following 2-hour folate deprivation.

Mark Mattson: Wow! That's a very rapid increase in homocysteine. Makes you wonder what might be happening in the brain under various conditions of changing diet, stress or injury.

Paul Aisen: Gladys, perhaps this is related to renal function.

Gladys Maestre: Paul, we explored that with creatinine levels and did not find any differences between genders.

Paul Aisen: But serum creatinine may not be an adequate indicator of glomerular filtration rate.

Eugene Rogers: Cystatin C will soon replace creatinine as a better indicator of renal clearance.

Gladys Maestre: Eugene, will look for that....

Andrew McCaddon: Gladys, I expect one reason it increases with age is because of the effects of oxidative stress on intracellular B12 metabolism.

Gladys Maestre: Andrew, exactly what do you mean?

Andrew McCaddon: Anyone here heard of glutathionylcobalamin? This is the obligate intracellular intermediate of B12 metabolism, and requires reduced glutathione for its synthesis. So, not only will oxidative stress impair the methionine synthase reaction, it will prevent the intracellular reduction of the vitamin to its active state, too!

Tom Shea: Definitely. We did see increased glutathione in both normal and ApoE knockout CNS following 1 month of folate deprivation, but only the ApoE knockout showed increased thiobarbituric acid reagent substances (TBARS, markers of antioxidant activity. Normal mice seem to be able to handle the problem. Makes me think so much is masked in normal aging.

Larry Sparks: Are homocysteine levels increased in any of the transgenic mouse models of AD?

Mark Mattson: Larry, we did not observe an increase in blood homocysteine levels in the line of AβPP mutant mice we have been studying (Dave Borchelt's; Swedish mutation). We have not looked at brain levels yet.

Tom Shea: We did see increased blood homocysteine in our mice fed folate-deprived diet.

Larry Sparks: Has anyone ever shown accumulation of homocysteine in senile plaques?

Mark Mattson: Larry, also, the increase in blood homocysteine levels in AβPP mutant mice on a folate-deficient diet was of a similar magnitude to that of non-transgenic mice on the folate-deficient diet.

Larry Sparks: Mark, any differences in cognitive ability or lack thereof induced?

Mark Mattson: Larry, yes. A folate-deficient diet exacerbates the spatial learning deficits in the AβPP mutant mice.

Larry Sparks: Mark, did the folate diet produce the same effect in the non-transgenic mice, but to a lesser extent?

Gladys Maestre: Mark, could you reverse those learning deficits with folate?

Mark Mattson: Gladys, we have not yet tried to reverse the learning deficits.

Andrew McCaddon: Regarding AβPP--my whole interest in this started when we found low B12 levels in the 717AβPP mutation family. I gather this mutation is also associated with low intracellular glutathione, so this fits in nicely.

Gabrielle Strobel: Tom, thanks for your comment to our chat page. Dave Holtzman has PDAPP x ApoE knockout mice that express individual human ApoE alleles. Would it make sense to test the E2, E3, and E4 mice in your folate diet/iron challenge paradigm to get at in-vivo mechanisms of the human genetic ApoE4 risk?

Tom Shea: Definitely.

Gabrielle Strobel: Mark, would such mice be interesting in your experiment with DNA repair and Aβ toxicity?

Mark Mattson: Gabrielle, studies of DNA repair and Aβ toxicity in the AβPP-mutant mice are underway. We eventually plan to cross the AβPP-mutant mice with mice that have altered DNA repair (knockouts and transgenics for specific DNA repair proteins).

Anthony White: Mark, did you look at the effect of folate deficiency in AβPP-mutant mice in old age of your mice (about 18 months), but before amyloid deposition? Just wondering about aging effects in these mice.

Mark Mattson: Anthony, we only observed hippocampal neuron loss in mice on a folate-deficient diet in AβPP-mutant mice in which amyloid deposition was beginning (8-10 months) or extensive (15 months).

Anthony White: Mark, was the cell loss associated with amyloid deposits or could it be related to aggregated but not 'deposited' amyloid?

Mark Mattson: Anthony, the cell loss first occurs at a time when Aβ is beginning to be deposited, but prior to the presence of overt plaque-like deposits. I believe the peptide is most toxic during the process of aggregation, and once deposited, its toxicity decreases. We really do think the peptide generates radicals, by a mechanism that requires iron or copper ions.

Anthony White: Mark, I agree. Was there any damage elsewhere in the brain or just hippocampal? Perhaps more sensitive measure of cell dysfunction should be examined.

Mark Mattson: Anthony, we have no data on other brain regions. The analyses have to be done using stereological counting methods, which are more difficult to apply to (e.g.) cortex.

Paul Aisen: Mark, based on your work, do you think homocysteine reduction would slow progression of AD?

Andrew McCaddon: Haven't done a formal clinical trial yet, but my personal opinion after looking at this for the last 10 years now is that correcting the hyperhomocysteinemia in our patients by folate and B12 supplementation doesn't reverse cognitive decline, though it might slow the process slightly.

Andrew McCaddon: One curious thing regarding the folate link--although serum folate is low, red cell folate isn't low in our studies, and none of the patients have any evidence of anemia--nor does the cognitive impairment precede anemia.

Paul Aisen: Andrew, I think it is clear that within normal ranges of folate, there is a negative association between folate level and homocysteine. So if homocysteine is neurotoxic, it would not be surprising to see cognitive effects without hematologic effects.

Andrew McCaddon: True Paul, but why is red cell folate normal? I wonder if the low folate is secondary to a functional B12 deficiency, resulting in a failure to polyglutamate intracellular folate, and a consequent loss of folate in urine.

Paul Aisen: Andrew, I suspect that the simplest explanation--that the main determinant of folate levels is dietary--is most likely. We are just beginning to look at this in an AD population.

Eugene Rogers: Andrew, serum folate may reflect more acute exposure to dietary folate as opposed to red cell levels, which are more of a long-term--and perhaps better--indicator of functional folate deficiency.

Monique Breteler: I don't know about deficiencies, but we clearly did not see a relation with dietary intake.

Gabrielle Strobel: Monique, is your comment in response to my earlier question about ApoE genotypes?

Monique Breteler: No, about folate intake. I don't know about ApoE genotype at this moment.

Gladys Maestre: Monique: We also did not see a relation with dietary intake, even though we went to every home to obtain dietary information.

Gabrielle Strobel: Gladys and Monique, what do you mean with no relation to dietary intake? No matter how much folate you eat with your foods, your body regulates homocysteine levels independent of diet? I don't' understand.

Monique Breteler: Neither do I. Maybe the dietary intake data do not properly reflect what you get as far as folate is concerned? We don't have blood levels.

Amy B. Graves: Monique and Gabrielle, this is not so unusual. We see no relation between serum cholesterol and dietary intake from a food frequency survey in two studies in Florida, one in Caucasians and the other in African Americans.

Gladys Maestre: Gabrielle, it is hard to really know anyone's dietary intake, but we look at this issue very carefully and still, folate and vitamin B12 levels do not correlate very well with intake. If you give a supplement, it is somehow different.

Andrew McCaddon: Just to throw some other things into the dietary discussion, we found that body mass index did not differ between our AD patient and control group. Not a wonderful nutritional status marker I know, but the best we could come up with!

Amy B. Graves: Andrew, body mass index in prevalent cases may not be useful.

Mark Mattson: Andrew, based on our animal models and Richard Mayeux's data, we think high calorie intake will increase risk of AD. However, BMI is not a direct (or even good) measure of calorie intake.

Andrew McCaddon: Regarding BMI, I know. It was the best we could do in the circumstances, though we did measure retinol binding protein in the earlier study, and found no difference.

Andrew McCaddon: Paul, fair enough, but what do you then make of the Nun Study findings??

Paul Aisen: Andrew, do you mean the predictive value of folate?

Andrew McCaddon: Paul, I mean, in the Nun Study, all the participants had exactly the same dietary intake of folate

Mark Mattson: Andrew, we observe a consistent, modest but significant decrease in homocysteine levels in rats or mice maintained on a dietary restriction regimen.

Paul Aisen: Andrew, diet may be the most important determinant of folate levels, but need not be the only one.

Andrew McCaddon: Mark, could the reduction in homocysteine levels in animals on dietary restriction be explained by a reduction in oxidative stress? It has taken me some time to realize this, but I think the bottom line is that homocysteine is in fact an excellent marker of oxidative stress in neuronal and vascular tissue!

Tom Shea: That's an interesting thought, Andrew.

Andrew McCaddon: Thanks Tom. It does take a bit of a different stance on the whole thing doesn't it!?

Gabrielle Strobel: Andrew, is homocysteine specific to AD in any way, then, or does it apply equally to all diseases of aging?

Andrew McCaddon: Gabrielle, aging in general, though oxidative stress is increased in diseases like AD.

Anthony White: Mark, we find that copper (even background levels) dramatically increases neuronal damage in the presence of homocysteine in cortical cultures. Have you looked at this in hippocampal cultures? Could the amyloid be supplying a source of transition metal for homocysteine reduction leading to toxicity?

Mark Mattson: Anthony, we have not looked at this, but the experiment would be to see if a copper chelator is protective.

Monique Breteler: Anthony, how and where could you measure copper in humans?

Anthony White: Monique, we have routinely been measuring copper in animal tissues including brain, liver and serum using inductively coupled plasma mass spectrometry. This can easily be done on human samples.

Tom Shea: Anthony, any interaction with good old aluminum?

Anthony White: Tom, alas, we haven't looked at that. Probably should but it's a word that people don't always want to hear.

Tom Shea: I know!

Gabrielle Strobel: We agree there is enough research data to increase daily folate intake recommendations. To what levels, and do they vary for cardiovascular and dementia prevention? Does this even make sense in absence of a proven relationship between dietary intake and serum levels?

Paul Aisen: Gabrielle, I would argue that we need clinical trials.

Gabrielle Strobel: Is eating large amounts of folate safe? No such thing as overdosing on spinach pie, perhaps, but how about people binging on folate supplements?

Paul Aisen: Gabrielle, large amounts of folate can precipitate manifestations of B12 deficiency.

Monique Breteler: I disagree that we have evidence that increasing folate will help prevent dementia.

Gabrielle Strobel: Monique, do you think the epidemiology is insufficient? How about the recent Framingham study?

Monique Breteler: Very interesting observation, Framingham. But yes, we have no data suggesting that higher folate intake reduces risk.

Eugene Rogers: Paul, Andrew and others: In relation to the Nun Study, CSF folate is concentrated from blood across the choroid plexus about four-fold, and it appears that serum or red cell folate are not predictors of CSF folate, and CSF may be associated with development of AD. Do you think that pathology of this region could play a role?

Paul Aisen: Eugene, nonetheless, peripheral folate levels are related to risk of AD.

Andrew McCaddon: Paul, yes, I think folate does play a role, but as I say, I wonder if the whole mechanism we are seeing is simply the effects of oxidative stress on the methionine synthase reaction, and on the intracellular processing of B12.

Larry Sparks: Andrew, do you think that one might find homocysteine in the vasculature of an individual with multiple sclerosis, then? I am asking because free-radical activity/oxidative stress in the vasculature can lead to inflammation, as we have shown for AD and in the brains of cholesterol-fed rabbits, and multiple sclerosis is a vascular inflammation disorder.

Andrew McCaddon: I haven't looked at multiple sclerosis personally, but probably the answer would be yes.

Larry Sparks: Thanks!

Gabrielle Strobel: Larry, are you measuring folate levels in your trial?

Larry Sparks: No, but many of the apolipoproteins and Aβ.

Gabrielle Strobel: Monique: What sort of trial do you want to see to find out if increased folate protects?

Monique Breteler: Prevention trials--not in MCI or demented people, but in non-demented.

Mark Mattson: I agree that prevention trials are critical, as trials in symptomatic patients are likely to have only a weak beneficial effect.

Gabrielle Strobel: Who would fund the Rolls Royce of trials for something as cheap as folate?

Gladys Maestre: Gabrielle, I think it is very important for clinicians to realize that it is worthwhile assessing homocysteine in a healthy subject and to take the opportunity to make a nutritional assessment. One of the problems is that indeed there are not clear cut "normal levels" for any of these three elements, by age stratum and gender! So what to treat is not clear.

Paul Aisen: Gabrielle and others, prevention trials are attractive in theory, but vastly more expensive to conduct.

Gabrielle Strobel: Is prevention the key with folic acid supplementation? I am asking because initial treatment trials for NSAIDs and estrogen have failed perhaps because the patients were too advanced in their disease. Do you think this would not happen with folate?

Paul Aisen: Gabrielle, but a trial of vitamin E did not fail ( Sano et al, 1997).

Larry Sparks: If you want to do a prevention trial based on increasing folate in the appropriate compartment (where it will do the most good) you must have a consistent means to increase that pool. Also, I believe that the direction with prevention trials will be that one will have to show first that the agent to be tested is effective as a treatment--at least in mild-to-moderate AD.

Tom Shea: It was only in culture, but when we increased folate to 5 times the normal culture level, we blocked Aβ-induced oxidative stress.

Anthony White: Tom, did that alter homocysteine levels in the cells/medium?

Tom Shea: Did not check, Anthony. Should have, will do…

Andrew McCaddon: If anyone wants to test the hypothesis, it would require culturing neurons in homocysteine-free medium, oxidatively stressing them, and measuring whether they export homocysteine into the medium. If anyone here can do that, I'd be grateful to know what they find!

Tom Shea: Actually, Anthony, trouble there: we have undetectable levels of homocysteine (by HPLC) unless we deprive folate for a few hours, so may not be able to determine. Any ideas?

Anthony White: Tom, there may be other means of measurement but not sure at present.

Andrew McCaddon: I saw a reference of a new assay that could detect nanomoles of reduced, free oxidized, protein-bound, and total homocysteine in human plasma (Chwatko et al. 2002.)

Tom Shea: I'll check that out.

Paul Aisen: Andrew, any thoughts on reduced folic acid as a supplement?

Andrew McCaddon: Re: reduced folate--not sure Paul. As you can tell, I am very much a "B12 man!"

Gabrielle Strobel: To everyone who has to leave: Thanks indeed for joining us today. Be back!

Mark Mattson: All: I'm heading back to the bench now--to get experiments we talked about here done while they're fresh in my mind. Have a good day.

Gabrielle Strobel: Thank you Mark! Good-bye for now.

Larry Sparks: Goodbye to all, and see you at the next meeting, Monique.

Monique Breteler: Bye, everybody.

Gladys Maestre: Bye, all.

Anthony White: Tom, have you looked at the redox status of the cultures treated with high folate levels? This may be related to Andrew's theory.

Tom Shea: Anthony: No, another thing that should be done.

Ralph Green: {enters}

Andrew McCaddon: Hi Ralph!

Gabrielle Strobel: Hi Ralph, you have joined us at the end of our hour, I suppose the announcement must have been confusing. Apologies; it said noon - 1 pm.

Ralph Green: Sorry I missed the discussion. The irony of getting the time wrong on an Alzheimer's chatroom strikes me. Perhaps I should get my homocysteine level checked!

Andrew McCaddon: Ralph, I hope you are keeping well. It's been a long discussion, and we covered some of the stuff I talked to you about in Sorrento last year.

Ralph Green: Hi Andrew.

Andrew McCaddon: There is another implication of our idea, namely that the currently available forms of B12 might not be the best to use in neuropathologic or vascular disease. Glutathionylcobalamin might be preferable. Have you had a chance to do any more work on glutathionylcobalamin, Ralph?

PS: How does the layperson get glutathionylcobalamin? I'm looking for a last-ditch try on slowing my mother's AD.

Andrew McCaddon: I'm afraid glutathionylcobalamin is not yet commercially available.

Ralph Green: Don Jacobsen is knowledgeable about GS Cbl.

PS: Not much hope, then. She no longer remembers family and is just now having eating difficulty. Anything promising? From what I gather, prevention is the best and intervention at my mother's stage is very poor or nil? Correct?

Andrew McCaddon: Dear PS, I am sorry to hear this. You could try some glutathione supplements like N-acetylcysteine or lipoic acid, but speak to your general practitioner first.

Ralph Green: This may date me but I'm going to say that this is the first chat room I've participated in, so please bear with my ineptitudes.

Andrew McCaddon: Ralph, it was fairly new to me an hour or so ago, too, but good fun!

Gabrielle Strobel: I have to run pick up my kids at school. But please do continue as long as you like, and thanks so much for joining us. Goodbye for now.

Andrew McCaddon: Anthony, you still here? I bet that cup of tea is cold now!

Anthony White: Andrew, I am still here. Yes, the tea is cold but the conversation is still hot.

Tom Shea: Andrew, N-acetylcysteine did compensate for folate deficiency in our little cultures.

Andrew McCaddon: Tom, yes, that is interesting. I've had some good results with patients given B12/folate/and NAC, but only case studies at present.

Tom Shea: Very interesting: if the manuscript comes back trashed, I'll suggest you as a reviewer next round!

Anthony White: PS, there are a number of treatments in clinical trials that are perhaps second-generation drugs. Not great but may offer some hope in the near future at slowing things down.

Andrew McCaddon: Ralph: I'll go and get us a pint. :-)

Tom Shea: A bit early for us in New England...... but looks good.

Andrew McCaddon: Warm beer beats Anthony's cold tea!

Eugene Rogers: Just got back in and missed some. Did anyone discuss the possible benefits of specific methionine cycle enzyme inhibitors on homocysteine reduction?

Ralph Green: Andrew: Having missed most of the action, I'm inclined to sign off now, even though I'm enjoying the experience immensely.

Andrew McCaddon: Ralph, before you go, any news on when and where the next homocysteine get-together is?

Ralph Green: I'm told that it has been changed to Berne or Basel (originally was going to be South Africa).

Andrew McCaddon: That sounds more convenient for us Europeans! Cheers.

Eugene Rogers: Interestingly, NAC has been used clinically for years (Mucomist) to treat acetaminophen overdose, which exerts toxicity via depletion of cellular glutathione.

Andrew McCaddon: Hi Eugene. Yes, that was our rationale for using NAC--we wanted to increase glutathione and B12 in our patients.

Ralph Green: I can see how this can become addictive but I really need to sign off now --its been fun and nice to meet all of you.

Andrew McCaddon: Anthony, could you do any of the tissue culture stuff I mentioned??

Anthony White: I am paid for prion work these days but could look at it. I suggest you try Mark or Tom, but failing that I could help. It does sound very interesting and I would like to help.

Tom Shea: Andrew, I have to get running, but would like to discuss more. Wonderful to talk with all of you.

Andrew McCaddon: OK Tom, will drop you a line. Got to go myself now. I've been left in charge of the children, and it's teatime here. Nice to chat with you all. Best wishes.

Anthony White: Yes, time I was going. Will keep in touch, it's been good talking to you all.

Background

Background Text
By Gabrielle Strobel

Now famous for having been overlooked since the 1960s as risk factor for heart disease, the amino acid homocysteine has in past few years become a suspect in neurodegenerative diseases, as well. Folate and vitamin B12 are required in the methylation of homocysteine to methionine, and extreme vitamin B12 deficiency has long been known to cause a dementia that is treatable with vitamin supplementation. Epidemiological research has implicated elevated levels of homocysteine in stroke (Ridker et al., 2000; Morris et al., 2000; Aronow et al., 2000), Alzheimer's (McCaddon et al, 1998), and, just this month, silent white matter infarcts (Vermeer et al. 2002).

But the relationship to dementia in general, and Alzheimer's in particular, has remained vague. There were negative studies as well as positive ones, (see for example Fallon et al., 2001). The correlation with Alzheimer's disease was weak (Breteler, 2000). Research on Parkinson's focused mostly on levodopa-induced increases of homocysteine, and no one knew precisely how this amino acid might promote neurodegenerative disease.

That's changing now. Two current studies have simultaneously strengthened the epidemiological evidence and advanced an intriguing cellular mechanism, providing ample fodder for a discussion (see ARF news story and comments). For one, the Framingham cohort has revealed a robust link between elevated homocysteine levels and increased risk of developing Alzheimer's. For another, a recent study from Mattson's lab (Kruman et al., 2000) began establishing a mechanism by showing that homocysteine increases hippocampal neurons' vulnerability to excitotoxic and oxidative injury in cell culture and in vivo. Mattson and Kruman's current paper (Kruman et al., 2002) enters amyloid into this scenario by suggesting that homocysteine makes hippocampal neurons particularly sensitive to Aβ-induced cell death, and that a diet low in folic acid promotes neurodegeneration in AβPP-transgenic mice.

The surprising twist in this amyloid connection is that homocysteine does not do its damage by increasing Ab production, as do the AbPP and presenilin mutations that underlie familial Alzheimer's disease. Rather, it impairs the neuron's ability to repair DNA damage. A gradual erosion of the cell's ability to repair DNA-damaged by reactive oxygen species and other assaults-has long been thought to be one mechanism of cellular aging. This raises the question whether homocysteine might be a player in neurodegenerative processes in general, acting via Aβ to exacerbate Alzheimer's and perhaps via different neurotoxins in other neurodegenerative diseases. Intriguingly, folate deficiency also worsens pathology in a mouse model of Parkinson's (Duan et al., 2002), though a molecular mechanism is unknown in this system.

Such data generate a sense of urgency about considering public health measures. Unlike other research implicating novel targets for which drugs must yet be developed, we could affect people's homocysteine levels via the food supply immediately.

Mark Mattson, who will lead the discussion, has posed the following questions for the online chat:

  • What are the implications of the link between homocysteine and risk for AD and PD, and genetic factors that may promote or prevent AD and PD?
  • What other epidemiological, clinical and basic research studies should be pursued to better clarify the role of homocysteine in neurodegenerative disorders?
  • Beyond folic acid, what dietary factors influence homocysteine levels?
  • What are the mechanisms whereby homocysteine promotes neuronal degeneration?
  • What are the contributions of effects on the cerebral vasculature versus direct effects on neurons?
  • What kinds of clinical trials should be done (primary prevention and treatment)?
  • What are the implications for education of the general public as to steps they can take to reduce risk of AD and PD?

The Alzheimer Research Forum invites you to consider these questions and join the debate. We also welcome your comments and will post them in advance of the debate to stimulate further thought.

Comment from Thomas Shea—Posted 28 April 2002.
Shea and colleagues have demonstrated that folate and vitamin E can compensate for the diminished oxidative buffering capacity of brains of apolipoprotein E-deficient mice. Normal and ApoE homozygous "knockout" mice were maintained for one month on a diet either lacking or supplemented with folate, vitamin E or iron as a pro-oxidant, after which brain tissue was harvested and analyzed for for thiobarbituric acid-reactive substances (TBARs) as an index of oxidative damage. Normal mice exhibited no significant difference in TBARs following iron challenge in the presence or absence of vitamin E, folic acid or both. Similarly, ApoE knockout mice exhibited no significant differences following dietary iron challenge in the presence or absence of vitamin E.

However, ApoE knockout mice accumulated significantly increased TBARs following iron challenge when folic acid was withheld, and accumulated even more TBARs when both folic acid and vitamin E were withheld. These findings demonstrate that ApoE knockout mice during vitamin deficiency are less capable of buffering the consequences of dietary iron challenge than are normal mice. Since the apolipoprotein E4 allele, which exhibits diminished oxidative buffering capacity, is linked to Alzheimer's disease (AD), these data underscore the possibility that critical nutritional deficiencies may modulate the impact of genetic compromise on neurodegeneration in AD.

Thomas B Shea, Ph.D.
Professor of Biological Sciences and Biochemistry
Director, Center for Cellular Neurobiology & Neurodegeneration Research
University of Massachusetts-Lowell

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Comments on this content

  1. I cannot accept the statement in the introduction that the association between elevated homocysteine levels and Alzheimer's disease is weak. An odds ratio of 4.5 was found for histopathologically-confirmed AD in our case-control study (Clarke et al, 1998). This study showed that patients with AD who had high homocysteine at baseline showed a more rapid progression of the disease than those with low homocysteine over the following three years. Furthermore, in a community-dwelling non-demented elderly cohort, cognitive scores were inversely related to the level of homocysteine (Budge et al., 2000). The prospective Framingham study referred to in the introduction shows an association between elevated homocysteine and AD that is as high, or higher, than found in prospective studies on its association with heart disease and stroke. An editorial published this month reviews the association between cognitive deficit in the elderly, elevated homocysteine and low levels of folate and vitamin B12 (Smith, 2002).

    References:

    . Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol. 1998 Nov;55(11):1449-55. PubMed.

    . Plasma total homocysteine and cognitive performance in a volunteer elderly population. Ann N Y Acad Sci. 2000 Apr;903:407-10. PubMed.

    . Homocysteine, B vitamins, and cognitive deficit in the elderly. Am J Clin Nutr. 2002 May;75(5):785-6. PubMed.

References

News Citations

  1. Down and In—the Location of Amyloid-β Does Matter

Webinar Citations

  1. Vascular Disease, Parkinson's, and Now Alzheimer's—Is Homocysteine the New All-Around Bad Guy?

Other Citations

  1. chat on cell cycle in AD

External Citations

  1. Ridker et al., 2000
  2. Morris et al., 2000
  3. Aronow et al., 2000
  4. McCaddon et al, 1998
  5. Vermeer et al. 2002
  6. Fallon et al., 2001
  7. Breteler, 2000
  8. Kruman et al., 2000
  9. Kruman et al., 2002
  10. Duan et al., 2002
  11. Bazzano et al., 2002
  12. McCaddon et al., 2002
  13. Clarke et al., 1998)
  14. see abstract
  15. Ho et al., 2002
  16. Sano et al, 1997
  17. Chwatko et al. 2002

Further Reading

Papers

  1. . Destabilization of beta-catenin by mutations in presenilin-1 potentiates neuronal apoptosis. Nature. 1998 Oct 15;395(6703):698-702. PubMed.